Tumor reactive T lymphocytes specifically recognize and lyse tumor cells based on the binding of a tumor reactive T cell receptor (TCR) with a specific peptide-MHC complex displayed on the surfaces of tumor cells. Nearly all of the peptides synthesized at this core facility have represented potential T lymphocyte epitopes restricted by either class I or class II MHC molecules derived from tumor-associated proteins. In several recent clinical trials in the Surgery Branch of the NCI, tumor reactive TCRs were cloned into retroviral vectors. These vectors were then used to modify genetically non-reactive T lymphocytes, and after transduction, the T cells were able to recognize specifically and lyse tumor cells in vitro. Furthermore, gene-modified T cells expressing tumor reactive TCRs mediated clinically objective regression of cancer in several patients after adoptive transfer. In order to extend these studies to patients with different types of cancer, we need to identify new tumor reactive TCRs that specifically bind to peptide-MHC complexes on cancer cells. Toward this aim, we have undertaken several studies to identify and clone such TCRs from HLA-A2 transgenic mice. In particular, these mice were immunized with peptides derived from the human proteins that represent potential tumor associated antigens including NY-ESO-1 and Carcinoembryonic antigen (CEA). After several rounds of in vitro stimulation, splenocytes from immunized mice specifically recognized human tumors expressing HLA-A2 and the full length protein. We cloned the reactive TCRs from these splenocytes and introduced them into non-reactive human T cells by means of RNA electroporation. The gene-modified human T lymphocytes then specifically recognized and lysed human tumor cells. We are currently trying to identify similar TCRs that could specifically recognize peptides derived from additional candidate antigens including mammaglobin-A, MAGE-A3, and MMP11.